The overall goal of this project is to discover novel anticancer drugs from endophytic fungi (fungi that live in the intercellular spaces) of desert plants with the broad long term objective of creating a library of this unexploited source of natural products for future drug discovery programs. The specific aims of the research to be undertaken will include sampling of 30 diverse Sonoran Desert medicinal plants and grasses for endophytic fungi; separating individual fungi and storing these to create a "library of endophytic fungi of Sonoran Desert plants "; culturing 500 endophytic fungi selected from this library on the basis of their diverse taxonomy; processing these fungal cultures and screening the resulting extracts for anticancer activity; fractionating selected bioactive extracts to isolate pure compounds responsible for their observed biological activity; determination of the structure of each biologically active compound; large-scale production of up to 3 compounds with unique structures and novel mechanisms of action followed by evaluation of their in vivo anticancer activity in human tumor xenograft mouse models. The methodologies to be utilized involve the separation and culturing of endophytic fungi in suitable solid or liquid media; successive extraction of fungal cultures with ethyl acetate and n-butanol and screening the resulting extracts in anticancer bioassays. Traditional proliferation/survival assays using three sentinel human cancer cell lines NCI-H460 (lung), and SF-268 (CNS-glioma)} and two new functional assays that monitor the inhibition of angiogenesis and the induction of heat shock protein expression will be used for screening. Dereplication of active extracts will be accomplished by taxonomically identifying the producing fungi based on literature reports and by analyzing bioactive extracts by LC-MS. Bioactivity-guided fractionation of unique active extracts will be performed using solvent-solvent partitioning, gel filtration and chromatography (CC, MPLC, prep. TLC, and HPLC). Structure elucidation of bioactive compounds will be achieved by a combination of spectroscopic (IR, UV, MS, and NMR) techniques, chemical manipulations, or X-ray crystallography as appropriate. Large-scale production (10-20 mg) of up to 3 selected compounds will be undertaken to obtain material for in vivo anticancer activity testing in hormone supplemented SCID mice bearing MCF-7 and T47D human tumor xenografts. We expect that having access to this unique source of bioactive natural products, combined with the use of both conventional and functional in vitro bioassays, followed by in vivo testing will lead to an efficient discovery of new anticancer agents which can be developed into clinically effective drugs with activity against solid tumors.